Process for preparing optically active beta-aminocarboxylic acids from racemic n-acylated beta-aminocarboxylic acids

ABSTRACT

A process is described for preparing optically active β-atninocarboxylic acids from racemic N-acylated β-aminocarboxylic acids by cnantiosclccthc hydrolysis of the N-acylated β-aminocarboxylic acid in the presence of a hydrolase by way of biocatalyst, wherein the N-acyl substituent of the N-acylated β-aminocarboxylic acid (I) exhibits Structure I in which R 1 , R 2  are each selected, independently of one another, from H, halogen, alkiyl residues, OH, alkoxy residues and aryloxy residues; R 3  is selected from halogen, alkoxy residues and aryloxy residues; (II) Structure IIA or IIB or the structure of the corresponding salts or (III) Structure III or the structure of the corresponding salt.

The invention relates to a process for preparing optically activeβ-aminocarboxylic acids.

Optically active β-aminocarboxylic acids occur in natural substancessuch as alkaloids and antibiotics, and their isolation is increasinglyattracting interest, not least on account of their increasing importanceas essential intermediate products in the preparation of medicaments(see, inter alia: E. Juaristi, H. Lopez-Ruiz, Curr. Med. Chem. 1999, 6,983-1004). Both the free form of optically active β-aminocarboxylicacids and their derivatives show interesting pharmacological effects andcan also be employed in the synthesis of modified peptides.

Until now the classical resolution of racemates via diastereomeric salts(proposed route in: H. Boesch et al., Org. Proc. Res. Developm. 2001, 5,23-27) and, in particular, the diastereoselective addition of lithiumphenylethylamide (A. F. Abdel-Magid, J. H. Cohen, C. A. Maryanoff, Curr.Med. Chem. 1999, 6, 955-970) have been established as methods for thepreparation of β-aminocarboxylic acids. The latter method is regarded ashaving been intensively researched and is preferentially adopted,despite numerous disadvantages that arise in the process. On the onehand, stoichiometric quantities of a chiral reagent are required, whichrepresents a great disadvantage in comparison with catalyticasymmetrical methods. Furthermore, expensive and, moreover, hazardousauxiliary substances such as, for example, n-butyllithium are requiredfor activating the stoichiometric reagent by deprotonation. Forsufficient stereoselectivity, in addition, the implementation of thereaction at low temperatures of about −70° C. is important, whichsignifies a high demand on the material of the reactor, additional costsand a high consumption of energy.

Although the preparation of optically active β-aminocarboxylic acids bybiocatalytic means plays only a subordinate role at the present time, itis desirable in particular by reason of the economic and ecologicaladvantages of biocatalytic reactions. The use of stoichiometricquantities of a chiral reagent is dispensed with, and small, catalyticquantities of enzymes, which constitute natural and environmentallyfriendly catalysts, are employed instead. In addition, thesebiocatalysts which are employed efficiently in the aqueous medium have,besides their catalytic properties and their high effectiveness, theadvantage—in contrast with a large number of synthetic metalliferouscatalysts—that the use of metalliferous feed materials, particularlythose which contain heavy metals and which are consequently toxic, canbe dispensed with.

In the state of the art there have already been numerous accounts of,for example, the enantioselective N-acylation of β-aminocarboxylicacids.

For instance, L. T. Kanerva et al. in Tetrahedron: Asymmetry, Vol. 7,No. 6, pp. 1707-1716, 1996 describe the enantioselective N-acylation ofethyl esters of various alicyclic β-aminocarboxylic acids with2,2,2-trifluoroethyl ester in organic solvents and lipase SP 526 derivedfrom Candida antarctica or lipase PS derived from Pseudomonas cepacia byway of biocatalyst.

V. M. Sánchez et al. investigated the biocatalytic resolution ofracemates of (±)-ethyl-3-aminobutyrate (Tetrahedron: Asymmetry, Vol. 8,No. 1, pp. 37-40, 1997) with lipase derived from Candida antarctica viathe preparation of N-acetylated β-aminocarboxylic ester.

In EP-A-0 890 649 a process is disclosed for preparing optically activeamino esters from racemic amino esters by enantioselective acylationwith a carboxylic ester in the presence of a hydrolase, selected fromthe group comprising amidase, protease, esterase and lipase, andsubsequent isolation of the unconverted enantiomer of the amino ester.WO-A-98/50575 relates to a process for obtaining a chiralβ-aminocarboxylic acid or its corresponding ester by bringing a racemicβ-aminocarboxylic acid, an acyl donor and penicillin G acylase intocontact under conditions for acylating an enantiomer of the racemicβ-aminocarboxylic acid stereoselectively, the other enantiomer beingsubstantially unconverted, thereby obtaining a chiral β-aminocarboxylicacid.

Desirable, in particular, would be the application to β-aminocarboxylicacids of a biocatalysis technology that is already practisedindustrially in the case of the α-aminocarboxylic acids. Of interest,above all, is the resolution of racemates of racemicN-acetyl-α-aminocarboxylic acids or corresponding derivativessubstituted on the N-acetyl group via enzymatic deacetylation usinghydrolases, in particular acylases. The racemic starting compounds caneasily be prepared with the aid of acetic-acid derivatives, and theirsynthesis is, in addition, possible in situ, so the N-acetylatedproducts can be employed directly in the biocatalytic reaction withoutan additional isolation step. The yields of acetylation reactions are inthe quantitative range, and the starting compounds, for instancechloroacetic acid, methoxyacetic acid or acetic anhydride, areinexpensive chemicals which are available in large quantities. A furtheradvantage of such acetyl derivatives in comparison with other acylderivatives is the easy separability of the N-acetylaminocarboxylic acidfrom the acetic acid (or the substituted derivatives thereof) after thereaction.

However, until now the application of this concept in respect ofβ-aminocarboxylic acids has failed. Unfortunately, it has turned outthat hydrolases, particularly acylases, do not appear to be suitable forreactions of such a type. H. K. Chenault, J. Dahmer, G. M. Whitesides,J. Am. Chem. Soc. 1989, 111, 6354-6364, established that neither acyclicnor cyclic N-acyl-β-aminocarboxylic acids are suitable as substrates.With regard to the acyclic compound, an N-acetyl compound wasinvestigated. This result has been confirmed by the inventors' ownexperiments with other hydrolases, in particular with acylases.

Until now there have been accounts only of the enantioselectivehydrolysis of racemic N-phenylacetyl-β-aminocarboxylic acids withpenicillin acylase (V. A. Soloshonok, V. K. Svedas, V. P. Kukhar, A. G.Kirilenko, A. V. Rybakova, V. A. Solodenko, N. A. Fokina, O. V. Kogut,I. Y. Galaev, E. V. Kozlova, I. P. Shishkina, S. V. Galushko, Synlett1993, 339-341; V. Soloshonok, A. G. Kirilenko, N. A. Fokina, I. P.Shishkina, S. V. Galushko, V. P. Kukhar, V. K. Svedas, E. V. Kozlova,Tetrahedron: Asymmetry 1994, 5, 1119-1126; V. Soloshonok, N. A. Fokina,A. V. Rybakova, I. P. Shishkina, S. V. Galushko, A. E. Sochorinsky, V.P. Kukhar, M. V. Savchenko, V. K. Svedas, Tetrahedron: Asymmetry 1995,6, 1601-1610; G. Cardillo, A. Tolomelli, C. Tomasini, Eur. J. Org. Chem.1999, 155-161). A disadvantage with this process is the difficultreprocessing of the product mixture after the enantioselectivehydrolysis. After separation of the free β-aminocarboxylic acid, amixture is obtained consisting of phenylacetic acid andN-phenylacetyl-β-aminocarboxylic acid, which is difficult to resolve.

Now the object underlying the present invention is to make available anew, simply and economically practicable process for preparing opticallyactive β-aminocarboxylic acids.

This object is achieved, surprisingly, by a process for preparingoptically active β-aminocarboxylic acids from racemic N-acylatedβ-aminocarboxylic acids by enantioselective hydrolysis of the N-acylated-βaminocarboxylic acid in the presence of a hydrolase by way ofbiocatalyst, wherein the N-acyl substituent of the N-acylatedβ-aminocarboxylic acid exhibits(I) Structure I

in which R¹, R² are each selected, independently of one another, from H;halogen, preferably chlorine, bromine and fluorine; alkyl residues withpreferably 1 to 10 C atoms, in particular methyl, ethyl, n-propyl,isopropyl, n-butyl and tert-butyl; OH; alkoxy residues with preferably 1to 10 C atoms, in particular methoxy and ethoxy, and aryloxy residueswith preferably 6 to 14 C atoms, in particular phenoxy; and

-   -   R³ is selected from halogen, preferably chlorine, alkoxy        residues with preferably 1 to 10 C atoms, in particular methoxy,        and aryloxy residues with preferably 6 to 14 C atoms, in        particular phenoxy;        (II) Structure IIA or IIB        or the structure of the corresponding salts or        (III) Structure III        or the structure of the corresponding salt.

Contrary to previous findings available from the literature and theinventors' own research results, quite unexpectedly a reaction of thespecial N-acylated β-aminocarboxylic acids with a hydrolase takes place.

The enantioselective hydrolysis proceeds in particularly effectivemanner with an N-acyl substituent having Structure I if R³ is chlorine,where appropriate R¹ or R² is also chlorine, or if R³ is methoxy or ifR¹, R² and R³ are each fluorine. Exemplary N-acyl substituents areN-chloroacetyl, N-dichloroacetyl, N-methoxyacetyl and N-trifluoroacetyl.A further advantage of these N-acyl substituents is that the acetic-acidderivatives arising therefrom in the course of hydrolysis can easily beseparated from the product mixture on account of their relatively lowmolecular weight.

In particular, the process is suitable for preparing optically activearomatic β-aminocarboxylic acids by conversion of an N-acylatedβ-aminocarboxylic acid having the following structure IV,

in which the N-acyl substituent is defined as previously; R⁴ is selectedfrom H; alkyl residues with preferably 1 to 10 C atoms, in particularmethyl, ethyl, propyl and butyl; OH, alkoxy residues with preferably 1to 4 C atoms, in particular methoxy and ethdxy; and halogen. It is aparticular advantage if the N-acyl substituent exhibits Structure I fromClaim 1, in which R¹ and R² are each H and R³ is chlorine, and R⁴ isequal to H.

The process according to the invention is particularly suitable forpreparing optically active 3-amino-3-phenylpropionic acid(β-amino-β-phenylpropionic acid) from the corresponding racemicN-acylated 3-amino-3-phenylpropionic acid.

The process according to the invention is also advantageous forpreparing optically active aliphatic β-aminocarboxylic acids byconversion of an N-acylated β-aminocarboxylic acid having the followingStructure V,

in which R⁵ stands for an alkyl group, in particular a methyl, ethyl,n-propyl, isopropyl, n-butyl or tert-butyl group, or a substituted alkylgroup, in particular a substituted methyl, ethyl, n-propyl, isopropyl,n-butyl or tert-butyl group. The substituents are preferably selectedfrom halogens, benzyl and N—, O— and S-containing substituents.

The racemic N-acylated β-aminocarboxylic acids employed as startingcompounds are generally obtained from the racemic β-aminocarboxylicacids by conversion with suitable acid chlorides or anhydrides. Alsopossible are the preparation of the racemic N-acylated β-aminocarboxylicacids in situ and their direct use in the biocatalytic reaction.

In the process according to the invention a large number of enzymes canbe employed as hydrolases; suitable hydrolases are, for example,acylases, proteases, lipases or esterases, preferably acylases. The useof pig-kidney acylase of type I has proved particularly suitable. Butthe reaction is also possible by using a protease, preferably derivedfrom Aspergillus, and more preferably from Aspergillus oryzae.

The enzyme that is used can be employed in native or immobilised form.The use of genetically engineered enzymes is also possible.

The process according to the invention is preferably implemented inaqueous solution. The pH value is usually between 6 and 10, preferablybetween 7 and 9.

In aqueous solution the concentration of the N-acylatedβ-aminocarboxylic acid preferably amounts to 2 to 40 wt. %, morepreferably 5 to 20 wt. %, relative to the total quantity in the reactionmixture.

Besides being carried out in aqueous solution, the process according tothe invention can also be carried out in organic solvents, preferably inwater-miscible solvents such as methanol and ethanol for instance, andalso in appropriate mixtures of organic solvents with water.

The quantity of enzyme to be added depends on the type of the hydrolaseand the activity of the enzyme preparation. The optimal quantity ofenzyme for the reaction can easily be ascertained by a person skilled inthe art by simple preliminary tests.

The hydrolysis of the N-acylated β-aminocarboxylic acid under enzymecatalysis is ordinarily carried out at temperatures between 10° C. and60° C., in particular between 20° C. and 40° C.

The progress of the reaction can easily be observed by conventionalmethods, for example by means of HPLC. The resolution of racemates issensibly brought to an end at a conversion of 50% of the racemicN-acylated β-aminocarboxylic acid. This is done, as a rule, by removingthe enzyme from the reaction chamber, for example by filtration,preferably ultrafiltration.

As a result of the enantioselective hydrolysis of the racemic N-acylatedβ-aminocarboxylic acid, the corresponding β-aminocarboxylic acid arisesfrom the one enantiomer, whereas the other enantiomer is substantiallyunconverted. The mixture that is now present, consisting of N-acylatedβ-aminocarboxylic acid and β-aminocarboxylic acid, can easily beseparated by conventional methods. Well-suited for the separation of themixture are, for example, extraction and/or filtration processes atsuitable pH values.

It is possible for the process according to the invention to be madestill more economical if, after separation of the desired enantiomer,the remaining, unwanted enantiomer is racemised in accordance withmethods known in the state of the art and is reintroduced into theprocess.

As a result of this recycling, it becomes possible to obtain a total ofmore than 50% of the desired enantiomer from the racemic N-acylatedβ-aminocarboxylic acid.

The process according to the invention is not only suitable forpreparing optically active β-aminocarboxylic acids but may also be partof complicated multistage syntheses, for example in connection with thepreparation of medicaments or crop-protection agents.

The invention will now be illustrated on the basis of the followingExamples.

EXAMPLE 1 COMPARATIVE EXAMPLE

In a reaction vessel a solution consisting of 900 μl of a 50 mMsodium-phosphate buffer with pH=8.0, 100 μl of a 0.1 M aqueous solutionof rac-N-acetyl-3-amino-3-phenylpropionic acid and 5 mg pig-kidneyacylase of type I (producer: Sigma) is stirred at 30° C. for 24 h andsubsequently the conversion-rate is determined by means of HPLC (column:Nautilus; eluent: H₂O and acetonitrile in a volume ratio of 80:20 with0.1 vol. % trifluoroacetic acid, 220 nm, 1 ml/min; injection: 900 μleluent+100 μl reaction mixture). The conversion-rate is <1%.

EXAMPLE 2

In a reaction vessel a solution consisting of 950 μl of a 50 MMsodium-phosphate buffer with pH=8.0, 50 μl of a 10% (w/vol. %) solutionof rac-N-chloroacetyl-3-amino-3-phenylpropionic acid in acetone and 5 mgpig-kidney acylase of type I (producer: Sigma) is stirred at 30° C. for24 h and subsequently the conversion-rate is determined by means of HPLC(column: Nautilus; eluent: H₂O and acetonitrile in a volume ratio of80:20 with 0.1 vol. % trifluoroacetic acid, 220 nm, 1 ml/min; injection:900 μl eluent+100 μl reaction mixture). The conversion-rate is 14%.

EXAMPLE 3

In a reaction vessel 50 ml of an aqueous solution consisting of apotassium-phosphate buffer with pH=7.0 and also 127 mgrac-N-chloroacetyl-3-amino-3-phenylpropionic acid (0.5 mmol) arecharged. Subsequently 120 mg of the pig-kidney acylase of type I(producer: Sigma) are added and the reaction mixture is allowed to reactat room temperature (about 25° C.). The conversion after five days is9%, and after 19 days 46% (according to HPLC of the reaction sample).

EXAMPLE 4

In a reaction vessel 50 ml of an aqueous solution of 127 mgrac-N-chloroacetyl-3-amino-3-phenylpropionic acid (0.5 mmol), which wasadjusted by means of NaOH to pH=8.2, are charged and brought to atemperature of 30° C. Subsequently 120 mg of the pig-kidney acylase oftype I (producer: Sigma) are added and the reaction mixture is allowedto react at a reaction temperature of 30° C. After five days theconversion is 24% (according to HPLC of the reaction sample). After aperiod of 13 days the reaction mixture is firstly separated from theenzyme component by ultrafiltration. A clear filtrate is obtained, fromwhich the conversion-rate and also the enantioselectivity with respectto the optically active 3-amino-3-phenylpropionic acid that has beenformed are then determined. The conversion-rate is 35%, and eevalues >98% were ascertained for the enantioselectivity.

EXAMPLE 5

In a reaction vessel a solution consisting of 950 μl of a 50 mMsodium-phosphate buffer with pH=7.5, 50 μl of a 10% (w/vol. %) solutionof rac-N-chloroacetyl-3-amino-3-phenylpropionic acid in acetone, and 5mg protease derived from Aspergillus oryzae (producer: Sigma: proteaseXXIII) are stirred at 30° C. for four days and subsequently theconversion-rate is determined by means of HPLC as in Example 2. Theconversion-rate is 6%.

EXAMPLE 6 Optimnised preparation of (S)-3-amino-3-(phenyl)propionic acid

In a 100 mL reaction vessel 60.4 mgrac-N-chloroacetyl-3-amino-3-phenylpropionic acid (purity: >98%; 0.25mmol) are added to 12.5 ml water and adjusted with NaOH to a pH value ofpH 7.75. After this, 2.5 mL of a 0.001 M cobalt(II)-chloride solutionare added, topping-up is effected with 12.5 mL of a buffer solution (50mM phosphate buffer), and the solution that has arisen is brought to atemperature of 37.5° C. Subsequently 60 mg of the pig-kidney acylase oftype I (producer: Sigma) are added and the reaction mixture is allowedto react at a reaction temperature of 37.5° C. After one day theconversion is 43.2%, and after two days 48.7% (according to HPLC of thereaction sample). After this, the reaction mixture is separated from theenzyme component by ultrafiltration. A clear filtrate is obtained, fromwhich the enantioselectivity with respect to the optically active(S)-3-amino-3-phenylpropionic acid that has been formed is thendetermined. For the enantioselectivity, ee values of 99.0% wereascertained.

EXAMPLE 7 Optimised preparation of optically active3-amino-3-(2-thiophenyl)propionic acid

In a 100 mL reaction vessel 63 mgrac-N-chloroacetyl-3-amino-3-(2-thienyl)propionic acid (purity: 98.3%;0.25 mmol) are added to 12.5 ml water and adjusted with NaOH to a pHvalue of pH 7.75. After this, 2.5 mL of a 0.001 M cobalt(II)-chloridesolution are added, topping-up is effected with 12.5 mL of a buffersolution (50 mM phosphate buffer), and the solution that has arisen isbrought to a temperature of 37.5° C. Subsequently 60 mg of thepig-kidney acylase of type I (producer: Sigma) are added and thereaction mixture is allowed to react at a reaction temperature of 37.5°C. After one day the conversion is 49.2%, and after two days 50.0%(according to HPLC of the reaction sample). After this, the reactionmixture is separated from the enzyme component by ultrafiltration. Aclear filtrate is obtained, from which the enantioselectivity withrespect to the optically active 3-amino-3-(2-thienyl)propionic acid thathas been formed is then determined. For the enantioselectivity, eevalues >99.0% were ascertained.

EXAMPLE 8 Optimised preparation of optically active3-amino-3-(p-fluorophenyl)propionic acid

In a 100 mL reaction vessel 66.1 mgrac-N-chloroacetyl-3-amino-3-(p-fluorophenyl)propionic acid (purity:98.2%; 0.25 mmol) are added to 12.5 ml water and adjusted with NaOH to apH value of pH 7.75. After this, 2.5 mL of a 0.001 M cobalt(II)-chloridesolution are added, topping-up is effected with 12.5 mL of a buffersolution (50 mM phosphate buffer), and the solution that has arisen isbrought to a temperature of 37.5° C. Subsequently 60 mg of thepig-kidney acylase of type I (producer: Sigma) are added and thereaction mixture is allowed to react at a reaction temperature of 37.5°C. After one day the conversion is 32.9%, and after two days 45.6%(according to HPLC of the reaction sample). After this, the reactionmixture is separated from the enzyme component by ultrafiltration. Aclear filtrate is obtained, from which the enantioselectivity withrespect to the optically active 3-amino-3-(p-fluorophenyl)propionic acidthat has been formed is then determined. For the enantioselectivity, eevalues >95.0% were ascertained.

1. A process for preparing optically active β-aminocarboxylic acids fromracemic N-acylated β-aminocarboxylic acids by enantioselectivehydrolysis of the N-acylated β-aminocarboxylic acid in the presence of ahydrolase by way of biocatalyst, wherein the N-acyl substituent of theN-acylated β-aminocarboxylic acid exhibits (I) Structure I

in which R¹, R² are each selected, independently of one another, from H,halogen, alkyl residues, OH, alkoxy residues and aryloxy residues, R³ isselected from halogen, alkoxy residues and aryloxy residues, (II)Structure IIA or IIB

or the structure of the corresponding salts or (III) Structure III

or the structure of the corresponding salt.
 2. Process according toclaim 1, wherein the N-acyl substituent of the N-acylatedβ-aminocarboxylic acid exhibits Structure I, in which R¹, R² are eachselected, independently of one another, from H, chlorine, bromine,fluorine, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl,methoxy and ethoxy and R³ is selected from chlorine and methoxy. 3.Process according to claim 2, wherein R¹ and R² are each H and R³ ischlorine.
 4. Process according to claim 2, wherein R¹ is equal to H andR² and R³ are each chlorine.
 5. Process according to claim 2, wherein R¹and R² are each H and R³ is methoxy.
 6. Process according to claim 1,wherein R¹, R² and R³ are each fluorine.
 7. Process according to claim1,wherein the N-acylated β-aminocarboxylic acid is an aromatic N-acylatedβ-aminocarboxylic acid having Structure IV

wherein R⁴ is selected from H, alkyl residues, OH, alkoxy residues, andhalogen.
 8. Process according to claim 7, wherein the N-acyl substituentexhibits Structure I, in which R¹ and R² are each H and R³ is chlorine,and R⁴ is equal to H.
 9. The process according to claim 1, wherein theN-acylated β-aminocarboxylic acid is an aliphatic N-acylatedβ-aminocarboxylic acids of the following Structure V,

in which R⁵ stands for an alkyl group.
 10. Process according to claim 1,wherein the hydrolase is an acylase, protease, lipase or esterase. 11.Process according to claim 9, wherein the acylase is pig-kidney acylaseof type I.
 12. Process according to claim 9, wherein the hydrolase is aprotease derived from Aspergillus.
 13. Process according to claim 11,wherein the protease is derived from Aspergillus oryzae.